Sex disparities in influenza: A multiscale network analysis

Summary Sex differences in the pathogenesis of infectious diseases because of differential immune responses between females and males have been well-documented for multiple pathogens. However, the molecular mechanism underlying the observed sex differences in influenza virus infection remains poorly understood. In this study, we used a network-based approach to characterize the blood transcriptome collected over the course of infection with influenza A virus from female and male ferrets to dissect sex-biased gene expression. We identified significant differences in the temporal dynamics and regulation of immune responses between females and males. Our results elucidate sex-differentiated pathways involved in the unfolded protein response (UPR), lipid metabolism, and inflammatory responses, including a female-biased IRE1/XBP1 activation and male-biased crosstalk between metabolic reprogramming and IL-1 and AP-1 pathways. Overall, our study provides molecular insights into sex differences in transcriptional regulation of immune responses and contributes to a better understanding of sex biases in influenza pathogenesis.

INTRODUCTION
Sex-related differences shaped by multidimensional biological characteristics that define females and males exert considerable influence on the pathogenesis of various human diseases (Klein and Flanagan, 2016), including autoimmune diseases (Jacobson et al., 1997), cancers (Cook et al., 2009(Cook et al., , 2011Kim et al., 2018), and infectious diseases caused by diverse pathogens (vom Steeg and Klein, 2016;Fischer et al., 2015;Sawyer, 2012). Each infectious disease exhibits a distinct pattern of sex bias in the prevalence, intensity, and outcome of infections (vom Steeg and Klein, 2016;Giefing-Krô ll et al., 2015), as well as in the responses to antiviral drugs and vaccines (Klein, 2012;Morgan and Klein, 2019). For example, females in the human population as well as in other mammals have a higher fatality rate following exposure to influenza A viruses (IAV) (World Health Organization, 2010) and more robust antibody responses and adverse reactions after vaccination than males Fink et al., 2018;Potluri et al., 2019), while accumulating evidence suggests a male bias in COVID-19 mortality (Scully et al., 2020;Takahashi et al., 2020). Thus an in-depth understanding of sex differences in the pathogenesis of diseases and consideration in the rational design of prophylactic and therapeutic strategies represent an important step toward segmented medicine.
Sex differences in organisms are as old as the development of sex itself. Sexual reproduction has been reported in early eukaryotes, such as metazoa, fungi, amoebozoa, euglenozoa, or haptophyte (Speijer et al., 2015). According to Rhen, four mechanisms contribute to sexual differentiation: (i) convergent evolution of genetic differences between sexes, (ii) sex-limited or differential expression of autosomal loci, (iii) transgenerational epigenetic effects, and (iv) phenotypic plasticity for sexual traits (i.e., environmental influences on sexual development) (Rhen, 2007). The observed sex differences in disease pathogenesis have been primarily attributed to the differences in the innate and adaptive immune responses between female and male mammals. In both arms of immunity, the sexes differ in multiple aspects, including the detection of pathogen nucleic acids by pattern recognition receptors (PRRs) such as the toll-like receptors (TLRs), the number and activity of immune cells, and the production of cytokines and chemokines (reviewed in (Klein and Flanagan, 2016;Galligan and Fish, 2015)). These intricate differences in immune functions between sexes have a powerful impact on infectious disease pathogenesis. For example, an augmented response to pathogens in females allows better control and clearance of pathogens while promoting increased immunopathology (vom Steeg and Klein, 2016;Klein, 2012;Fischer et al., 2015). During influenza infections, female mice exhibited a more robust induction of pro-inflammatory cytokines and chemokines in their lungs, including TNF-a, IFN-b, IL-6, and CCL2, accompanied by greater weight loss, hypothermia, and mortality than male mice .
The etiology underlying sex differences in immunity involves intrinsic and extrinsic factors that exert a combinatorial effect on the immune system's functioning. Although the microbiome (Markle et al., 2013;Yurkovetskiy et al., 2014, Vom Steeg andKlein, 2017;Vemuri et al., 2019) and nutritional status (Khulan et al., 2012;Tobi et al., 2009;Sinha et al., 2003;Kawai et al., 2010;Osrin et al., 2005;Christoforidou et al., 2019) have been implicated in modulating immune responses, sex hormones and genetic mediators are the most widely appreciated factors shaping differential immunity between females and males. In addition to the profound effects of sex hormones that have been extensively demonstrated, genetic differences attributed to immune-related genes and microRNAs (miRNAs) that are located on the sex chromosomes also play an important role in determining the distinct immune responses between sexes, especially in prepubertal children, postmenopausal females, and age-matched males (reviewed in (Galligan and Fish, 2015;Klein and Flanagan, 2016;Fischer et al., 2015;Fish, 2008;Klein, 2000;vom Steeg and Klein, 2016;Schurz et al., 2019;Bianchi et al., 2012)). However, the pathways and cellular responses that mediate the differences in response to influenza infection have not been well elucidated. Moreover, the confounding effects of hormone and genetic factors in intact animals and human populations impose a challenge in resolving the mechanism of sex differences in influenza pathogenesis.
To address the molecular mechanisms underlying sex differences in influenza infection responses, we assessed the transcriptomic dynamics of blood cell responses in neutered female and male ferrets throughout the infection. We detected a temporal shift in mounting immune responses against viral infection between females and males. Using a multiscale network-based approach, we further identified pathways and cellular processes commonly induced in both sexes and those uniquely regulated in females or males. Our data revealed gene regulatory pathways that were differentially regulated between sexes and likely involved in marked sexual differences in influenza virus-induced pathogenesis, providing molecular and functional insights for functional evaluation and development of therapeutic strategies.
To evaluate the genetic factors-mediated sex differences in the immune response to influenza virus infection, we conducted a study with neutered female and male ferrets. Ferrets are considered the gold standard in influenza research (Skarlupka and Ross, 2020;Belser et al., 2011). The ferret is currently the only small mammalian model available that is equally well suited to studying both influenza pathogenesis and transmission. Ferrets and humans show very similar clinical and pathological signs of influenza infection, depending on the host age, viral strain, environmental conditions, degree of secondary bacterial infection, and other variables. Clinical features in both organisms involve fever, nasal secretion, coughing, gastrointestinal complications, serum abnormalities, weight loss, lethargy, lymphopenia, hypercytokinemia, and transmission to susceptible contacts (Belser et al., 2011).
Concerning cellular immune response, cytotoxic lymphocytes are the primary cells important for attacking the immune system against infected cells. In the case of the humoral immune response, IgA antibodies seem to protect the upper tract in both humans and ferrets (Maher and DeStefano, 2004). Overall, the ferret model has played an essential part in mapping out the pathogenesis of the typical influenza infection, transmission, and reassortant variants of the virus, Reye's syndrome, drug treatment, and resistance of the influenza disease to many antiviral drugs (Burlington et al., 1981;Herlocher et al., 2003).

An integrated network approach for systematic characterization of the immune response to influenza infection in females and males
We examined the transcriptional response of neutered female and male ferrets in blood cells over the course of the infection ( Figure 1A). Briefly, whole blood samples were collected from spayed adult female and castrated adult male ferrets infected with the influenza virus A/CA/07/2009 (H1N1pdm09) strain over a period of 1 to 8 (for males) or 1 to 14 (for females) days postinfection (dpi). Samples from infected animals and uninfected control samples, including those collected before the infection from infected animals or before and after infection from uninfected individuals, were used to serve as the baseline for the comparison of infection responses within each sex. The whole blood transcriptome was profiled to assess the impact of genetic sex on immune response regulation without the additive influence of sex hormones. Given the time frame of sample collection, this study primarily focused on identifying sex differences in innate immunity and the early phase of adaptive immunity (Rowe et al., 2010 To systematically characterize sex differences in the immune response to influenza virus infection, we used a coexpression network-based approach that integrated transcriptional patterns with physiological traits and enabled the identification of sex-specific gene expression signatures ( Figure 1B). Specifically, differential expression analysis was carried out within each sex by comparing the infected samples with the uninfected controls at each time point after infection. A Multiscale Embedded Gene co-Expression Network Analysis (MEGENA) was then performed (Song and Zhang, 2015) on the transcriptomic data for each sex separately. We further prioritized the co-expressed gene modules in each sex identified by MEGENA based on their enrichment for the respective sex-specific differentially expressed gene (DEG) signatures that captured molecular responses to influenza infection. We characterized these modules with the Molecular Signature Database (MSigDB) and blood cell-type-specific marker gene signatures. Disease-relevant modules were then correlated with pathological traits, including weight loss and viral load, and selected for comparison of response signatures and co-expression patterns between sexes. Key regulators determined by multiscale hub analysis (MHA, see STAR Methods), as well as module compositions and within-module correlation patterns in selected modules, were further investigated to elucidate shared or sex-unique processes and regulatory patterns.

The pathophysiological response during infection is significantly different between the sexes
We were first interested in the overall physiological responses, such as weight loss and viral load ( Figure S1) and potential sex difference during influenza infection. We observed inter-ferret variability and identified In the serial sacrifice study of female ferrets (n = 45), blood samples were collected at the uninfected baseline (n = 26) and days 1 (n = 10), 3 (n = 12), 5 (n = 8), 8 (n = 7), and 14 (n = 8) postinfection from animals sacrificed at a given day. For the standard study of female ferrets (n = 9), samples were collected at baseline and from naive (n = 4) and infected (n = 5) animals at days 3, 5, and 8 postinfection. Similarly, for the standard study of male ferrets (n = 34), samples were collected at baseline (n = 33) and at days 1, 3, 5, and 8 postinfection (n = 30). (B) Framework for systematic characterization of sex difference in immune response to influenza infection with whole blood transcriptome. Transcriptomic data generated from infected and control samples of each sex were used for differential expression analysis and Multiscale Embedded gene co-expression network analysis (MEGENA). Diseaserelevant modules of co-expressed genes were then identified by the enrichment of differentially expressed genes (DEGs) and the association with physiological traits. Key regulators in those selected modules were predicted using the adopted Fisher's inverse Chi-square approach in MEGENA (see STAR Methods). Pairwise comparisons were performed to further determine similarities in module composition, module key regulators, and intra-module connectivity (see STAR Methods).

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iScience 25, 104192, May 20, 2022 3 iScience Article significant differences in weight loss and viral load, using a 95% CI. Concerning the weight loss of infected ferrets, the sickest female maintained 77% of her original body mass after eight days ( Figure S1A). In comparison, the healthiest infected females kept their body mass at 97%. For males, we observed a slightly narrower range of weight loss. The sickest male lost weight by 22% after eight days, whereas the healthiest infected male maintained his weight at 94%. Weight loss at day 8 postinfection is significantly different between females (mean 87.8%) and males (mean 85.4%; p = 0.011; Figure S1A). With respect to viral load, we have observed the largest variation at 1 dpi. At this time point, females experienced a viral load with a range between 3.05 3 10 6 and 3.10 3 10 8 PFU/mL (mean 9.70 3 10 7 PFU/mL) and males with a range between 150 and 1.07 3 10 8 PFU/mL (mean 4.11 3 10 6 PFU/mL). The difference in viral load at 1 dpi between females and males is significant (p = 0.0011; Figure S1B). Overall, although females experienced a significantly larger viral load at 1 dpi, their weight loss during infection is less severe than the males'.
We further investigated the cell content of immune cells during infection. As we did not have measured immune cell data (e.g., from flow cytometry measurements), we utilized publicly available single-cell data. Owing to the limited availability of ferret single-cell information, in particular from ferrets infected with the influenza virus, we settled for a recently published dataset by Lee et al. from bronchoalveolar lavage fluid (BALF) of COVID-19 infected ferrets (Lee et al., 2021) (GEO: GSE171828). Although this was suboptimal, we hypothesized that influenza and COVID-19 had similar immune cell-type-specific transcriptional signatures in BALF and blood during the first few days after infection. However, a more appropriate, single-cell-based approach to this infection scenario is required for detailed analysis. With these limitations in mind, we deconvoluted the ferret bulk dataset with the Lee et al. single-cell data using MuSiC . The originally assigned cell types by Lee et al. included dendritic cells, macrophages, granulocytes, mast cells, natural killer (NK) cells, gd-T cells, CD8 + T cells, CD4 + T cells, proliferating T cells, B cells, plasma cells, and epithelial cells.
Overall, red blood cells are overly abundant in both female and male ferrets ( Figures S2A and S2C). But other cell types such as granulocytes and M1 macrophages are similarly abundant. We were further interested in the relationship between cell types and physiological traits. Most pronounced in both sexes are the negative correlations between weight and cell types, such as granulocytes, M1 macrophages, mast cells, plasma cells, and proliferating T cells ( Figures S2B and S2D). Granulocytes and macrophages are the first line of immune cell response. So are mast cells that identify viral threats through several different receptors, such as TLR3 or Rig-I (DDX58). Interestingly, these three cell types are strongly positively correlated with viral load in females but not as much in males. On the other hand, immune cells of the adaptive immune system, such as proliferating T cells or plasma cells, have strong negative correlation with weight in males and weaker correlation in females. Generally, females mount faster and more robust innate and adaptive immune responses than males. However, our temporal window only extends to 8 days (14 days for females), which does not cover the full extent of adaptive immune response. Thus, we identified a strong correlation between innate immune cells and viral load in females. On the other hand, we observed a quenched immune response by immune cells of the adaptive immune system. However, this restricted response of the adaptive immune system could also be attributed to the limited temporal window of sample selection, as a more robust adaptive immune response may occur at a later time.
We were further interested in the detailed temporal response of immune cells ( Figures S2E-S2H). Most noteworthy is the female response of dendritic cells compared to males. At 3 dpi, the relative abundance of dendritic cells in females is almost three times higher than in males (1.94 vs. 0.66%, resp., p = 1.11 3 10 À4 ). In contrast, dendritic cells in males remain almost constant during the entire course of infection. Berghö fer et al. discovered in healthy subjects that TLR7 ligands induce a higher IFN-a production in females (Berghö her et al., 2006). However, the authors did not differentiate cell abundance between the sexes. Thus, other processes induced by the viral infection may cause the increased production of dendritic cells in females compared to males. Although taken with caution as cell type abundance is relative and inferred by transcription-based single-cell deconvolution, overall, a higher relative abundance of immune cells was observed in females compared to males.

Differential expression analysis reveals global temporal differences in immune response dynamics between sexes
Following the recognition of viruses, various immune cells equipped with versatile strategies to mount a potent antiviral response are mobilized to establish physical and chemical barriers against viral infection. To determine the global dynamics of blood cell response in females and males over time, we first carried out differential expression analysis of the whole blood transcriptome data collected over eight-or 14-day periods after influenza infection in comparison with sex-matched uninfected controls. Among different sets of DEGs identified over time in females and males (Tables S1 and S2), the most significant overlaps between any two given sets were observed at 3 and 5 dpi within or between the sexes, followed by those detected between 3 to 5 dpi in each sex and 1 dpi in females or eight dpi in males (Figures 2A and S3). It is noteworthy that DEGs at 14 dpi in females did not share significant overlaps with any other sets iScience Article ( Figure 2A), which suggests a largely subsided initial immune response by 14 dpi. This finding is consistent with the reported kinetics of an early innate phase in biphasic immune responses during influenza infections in ferrets (Rowe et al., 2010). To further understand the biological processes associated with DEGs during viral infection, we explored the enrichment of MSigDB gene sets in the upregulated and downregulated DEG signatures at each time point over 8 days in each sex. As expected, immune response related pathways are enriched in the signatures in both sexes and they included the innate immune response and cytokine signaling -particularly type I (a=b) and II (g) interferon (IFN) signaling -hemostasis, the urokinase-type plasminogen activator (uPA) and its receptor (uPAR)-mediated signaling, DNA replication, and the cell cycle ( Figure 2B). These processes exhibited differential temporal kinetics of activation, with IFN signaling being initiated immediately upon infection. However, cell cycle-related processes were revealed later in the infection ( Figure 2B). We also detected enrichment of downregulated genes during infection in T cell functioning and IL-17 pathways in both sexes, and several generic cellular processes were related to transcription and translation in males ( Figure 2B, Tables S3 and S4).
To further investigate the temporal dynamics of infection-responsive processes in both sexes, we examined the fold change (FC) of DEGs associated with the enriched MSigDB gene sets over time. We found more robust activation of type I and II IFN signaling pathways in females than males, as many genes were immediately and more strongly activated at 1 dpi in females ( Figures 3A and 3B). A temporal shift in the expression kinetics between sexes was also evident in platelet activation, signaling, and aggregation, which were important in maintaining hemostasis and modulating inflammation (Le et al., 2015). Many genes involved in platelet activity were immediately activated at 1 dpi and substantially subsided by 8 dpi in females, in contrast to an attenuated activation at 1 dpi and continuous expression beyond 8 dpi in males ( Figure 3C), further suggesting a more rapid response in females than males. Interestingly, we also detected a DEG, GLIPR1L2 (GLI pathogenesis-related one like 2), with extreme temporal shift as manifested by inverse alteration kinetics between the sexes (r = À0.9993, p = 0.0238). GLIPR1L2 was downregulated at 1 to 3 dpi and returned to baseline expression by 5 dpi in the females but was continuously downregulated from 3 dpi onward in males ( Figure 3D). Although the function of GLIPR1L2 in viral infections remains unclear, it has been shown to reside in the same module with GLIPR1 (GLI pathogenesis-related protein 1) and GLIPR1L1 and can be targeted by the tumor suppressor p53 during cancer development and progression (Ren et al., 2006). Taken together, these results indicate temporal differences in the immune response dynamics between the sexes, marked by a prompt response in females compared with a lagged response in males.
Co-expression network analysis identifies shared and sex-unique expression patterns in the immune response Although differential expression analysis enabled a global overview of the immune response in both sexes over the course of the infection, it did not provide the resolution for gene sets with sex-unique expression patterns. To dissect the sex-biased factors, we next sought to employ a network-based approach to identify consistent and unique gene expression patterns between sexes with increased granularity. We performed MEGENA on the transcriptomic data corresponding to each sex (Tables S5  and S6) and prioritized the modules of co-expressed genes in each sex based on their enrichment of DEGs. To assess the direct link between modules and pathogenesis, we also examined correlation between modules and physiological features, including viral titers in the nasal washes and weight change ( Figure S1). Overall, we identified modules enriched for upregulated or downregulated DEGs, or both, at one or more time points in each sex ( Figures 4A, 4B, S4, Tables S7, and S8). Those modules were enriched for a diverse array of gene set signatures involved in distinct but related biological processes, such as IFN signaling, inflammation, immune cell migration, hemostasis, and apoptosis induction and clearance exerted by various blood cells ( Figure 5), revealing more specific processes than those associated with DEGs globally. Some modules were also positively associated either with viral load in one sex or both or with weight loss in males ( Figures 5 and S5). For example, viral load-related modules in both females and males were consistently enriched in the IFN signaling and primarily type I IFN signaling pathways. In contrast, viral load-related modules in females alone were associated with specific inflammation-related pathways, lysosome-related processes, the unfolded protein response (UPR), and hemostasis. Those only in males were associated with apoptosis-related pathways (Figures 5 and S5). Although we did not detect any weight loss-related modules in females, the weight loss-related modules in males were associated with generic processes and pathways such as transcription, translation4-1BB-dependent immune response and porphyrin metabolism (Figures 5 and S5 iScience Article between weight loss and underlying molecular processes. Moreover, several modules in each sex were enriched for blood cell-type-specific signatures: (i) the female modules M35, M196, and M524 and the male module M127 were enriched for the T cell signature which was associated with either the translocation of ZAP-70 or the immunological synapse, (ii) the male module M15 was enriched for the generation of second messenger molecules pathway, and (iii) the male modules M17, M134, M356, and M523 were enriched for the CD19 + B cell signature which was related to primary immunodeficiency (Tables S7  and S8). These results elucidate a comprehensive landscape of complex immune responses elicited by diverse types of immune cells. Although both sexes have a common arsenal of strategies as immune system response, females and males further employ different pathways and processes contributing to the fine granularity of sex disparity in such a host-pathogen system. We also investigated the temporal response of the co-expression networks. For this purpose, we constructed separate co-expression networks for each time point. We assessed the similarity (or difference) of modules between time points by using the Jaccard index, focusing on the gene content by disregarding the network connectivity. This allowed more robust temporal trajectories between modules with significant overlap measured by Fisher's exact test (FET). For our comparative analysis, we focused on overall common responses, such as the upregulated type I IFN signaling and the downregulated AHSP pathway. Figures S6  and S7 show the time-resolved module development of type I IFN signaling in females (relating to modules M104 and M400) and males (relating to modules M3, M26, M165, and M478), respectively. Figures S8 and S9 depict the AHSP pathway in females (relating to modules M414) and males (relating to modules M6, M60), respectively.
We observed a similar temporal shift between the sexes in type I IFN signaling module conservation as we observed with the gene expression of immune genes and corresponding functions. The female type I IFN response is established and conserved early between 1 dpi and 3 dpi ( Figures S6A and S6C), whereas males muster their IFN defense much later ( Figure S7A). Their IFN response pathway is organized at 3 dpi and 5 dpi ( Figure S7C), with an established network at 5 dpi continuing to eight dpi ( Figure S7D). However, the male IFN signaling network displayed an early organized phase between 0 dpi and 1 dpi but was disorganized at 3 dpi ( Figure S7A). On the other hand, interferon-induced helicase C domain 1 (IFIH1) is central in IFN signaling for both females and males. The overall downregulated AHSP pathway shows similar behavior ( Figures S8 and S9). We observed the strongest conservation in module membership and connectivity between 5 dpi and 8 dpi in males ( Figures S8A and S8D). On the other hand, females have a highly conserved temporal response (especially the interaction between hemoglobin subunit beta (HBB) and the novel gene ENSMPUG00000010158) across all time points ( Figure S9A).
To identify the molecular factors mediating sex differences in the immune response to infection, we performed an in-depth comparison of the structural and functional relationships between modules across the female and male networks. Given that some processes and pathways were consistently detected over the 8-day period in the female and male networks, we first asked if the modules enriched in the same gene set signatures in both sexes shared similar members and key regulators. These shared signatures depicting temporal dynamics of the immune response included (i) IFN signaling (primarily type I IFN), lysosome-related processes, hemostasis, mitotic cell cycle, and DNA replication, and graft versus host disease signature, which exhibited gradual activations over time, and (ii) primary immunodeficiency signature, AHSP pathway, and translocation of ZAP-70 to the immunological synapse, which were generally A B  Tables S5 and S6, and information about modules enriched in DEGs at each time point in Figure S4 and Tables S7 and S8. iScience Article suppressed in both sexes (Figure 4), similar to those observed in DEGs. We conducted pairwise comparisons between the modules in the female networks and those in the male one by using FET to determine the similarity of modules and their key regulators (Table S9). Not surprisingly, the modules enriched for the same signatures in both sexes had a significant overlap (Table S9). However, the key regulators in modules with matched signatures shared limited overlaps ( Figure S10 and Table S9). These results suggest similar transcriptional programs in both sexes upon infection are coordinated by different regulators in females and males.
To further pinpoint the sex-specific gene co-expression patterns that implicated distinct functional signatures, we next focused on the modules enriched for sex-unique gene signatures to determine the uniqueness of those modules to each sex. We examined module-module overlap by using FET and performed differential gene correlation analysis ( (Table S10). We identified one female module, M139, which was unique to females determined by FET (Table S9) and was associated with the activation of chaperone genes by XBP1(s) ( Figure 5 and Table S7). DGCA The modules and their hierarchy were shown on the right side of the plot and colored by sex. The asterisks and squares next to the MSigDB gene sets indicate the presence of correlation between at least one module among those associated with the corresponding MSigDB gene set and physiological traits (i.e., asterisks for viral load and squares for weight loss). Only the correlations with p value % 0.05 and coefficient R 0.5 were included. Detailed information can be found in Figure S5. Detailed time-resolved development of the IFN signaling modules and the AHSP pathway modules for females and males can be found in Figures S6 and S7, and in Figures S8 and S9, respectively. Detailed information about key regulators in modules can be found in Figure S10.

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iScience 25, 104192, May 20, 2022 9 iScience Article identified two male modules, the AP-1 (activator protein-1)-associated M152 and the IL-1-associated M569, which were specific to males (Tables S8 and S10; Figure 5). XBP1 (X-box binding protein 1) was the key regulator of the female-specific module M139 ( Figure 6A). The majority of the genes within the female module, M139, exhibited a rapid and strong induction immediately following infection at one dpi in females (Figure S11A) compared with a milder induction in males ( Figure 6B), suggesting a prompt and potent activation of the IRE1/XBP1 branch of the UPR (So, 2018;Hetz, 2012) during infections, specifically in females. The male-specific module M152 was enriched for later-activated DEGs, including two key regulators HPGD (15-Hydroxyprostaglandin dehydrogenase) and MS4A2 (the b chain of the high-affinity IgE receptor (FcεRIb)), which were downregulated at three dpi and upregulated at 5 to 8 dpi in both sexes ( Figures 6C, 6D, and S11B) and likely linked to FcεRI-mediated and prostaglandin E 2 (PGE 2 )-mediated inflammatory responses (Ugajin et al., 2011;Inage et al., 2014;Mkaddem et al., 2019;Rastogi et al., 2020;Tai et al., 2006). The other male-specific module, M569, enriched for the upregulated DEGs at 3 to 8 dpi in males ( Figure S11C) iScience Article contained two key network regulators, ACSL3 (acyl-CoA synthetase long-chain family member 3) and IL18-RAP (IL-18 receptor accessory protein) ( Figures 6E and 6F), implying a link between lipid metabolism and inflammation (Yan et al., 2015;Roelands et al., 2019;Remmerie and Scott, 2018;Batista-Gonzalez et al., 2020;Kobori et al., 2018;Yasuda et al., 2019). Taken together, these results revealed sex-dependent regulatory relationships and the extent of the modulation of genes involved in IRE1-XBP1, AP-1, and IL-1 pathways upon infection, suggesting sex differences in specific aspects of the UPR, lipid metabolism, and inflammation independent of sex hormones.

DISCUSSION
A comprehensive understanding of the molecular mechanism underlying sex differences in response to viral infections has been either largely limited to sex chromosome-linked genes or hindered by the presence of confounding factors, especially in human populations. In this study, we systematically characterized sex differences in the transcriptional landscape of blood cell responses to influenza virus infection in neutered ferrets, thus precluding the impact of other factors. We detected temporal and regulatory differences in the processes commonly induced in both sexes as evidenced by a more rapid response in females than males and different key regulators orchestrating those shared responses. With a network-based approach, we further identified sex-specific modules of co-expressed genes that exhibited unique regulatory relationships in each sex. Those sex-unique modules revealed a rapid UPR activation via the IRE1/XBP1 pathway upon infection in a female-biased manner and a male-specific regulation of inflammatory responses mediated by a cascade involving FcεRI, AP-1, and PGE 2 , and the IL-1 family signaling associated with lipid metabolism. These data provided a comprehensive picture of genetic factors-mediated molecular differences in the immune response to influenza infection between sexes.
Sex-differentiated gene expression in normal and disease states of human tissues has been identified in many studies (Kassam et al., 2019;Trabzuni et al., 2013;Lopes-Ramos et al., 2020;Bongen et al., 2019;Jansen et al., 2014). However, the interpretation of underlying molecular mechanisms of infectious disease pathogenesis, especially genetic factors-mediated baseline differences between the sexes, remains challenging because of the inevitable presence of confounding factors such as sex hormones, age, microbiota composition, and nutritional status in human studies. With the neutered adult ferrets, our study provided a unique opportunity to interrogate sex-biased transcriptional response to influenza virus infection driven by genetic mediators. From the blood transcriptomic data, we detected a temporal difference in the dynamics of immune responses between sexes, manifested by a more rapid activation in females than males upon infection. The temporal difference between the sexes was observed at the gene co-expression network level at different time points. The immediate response in females is likely attributed to germline-encoded bias in innate immune sensing, as TLR7 that is located on the X chromosome and encodes an innate PRR-recognizing single-stranded RNA can not only have a higher expression level in females than males because of X-inactivation escape (Pisitkun et al., 2006) but also induce more robust IFN-a production in peripheral blood mononuclear cells (PBMCs) isolated from females than those from males in vitro (Berghö her et al., 2006). The sex disparity in TLR7 expression may also relate to the observed difference in the response of dendritic cells. After cell type deconvolution using a public singlecell dataset, we identified a significant increase in relative abundance of dendritic cells in females at 3 dpi, whereas the dendritic cell abundance in males remained constant.
We further found a limited overlap in key network regulators modulating commonly altered processes upon infection between the sexes, suggesting differential regulation of various aspects of immune responses in females and males. This observation is consistent with a previous report of sex differences in regulatory targeting patterns identified across multiple healthy tissues in humans (Lopes-Ramos et al., 2020). We further identified a peculiar gene, GLIPR1L2, with an extreme temporal shift as manifested by inverse alteration kinetics between the sexes. Although its potential role during infection remains obscure, GLIPR1L2 is a target of tumor suppressor p53 during cancer development and progression (Ren et al., 2006).
Using a network-based approach, we identified sex-specific modules that represented distinct pathways differentially impacted by sex and exhibited unique regulatory structures and co-expression patterns. These modules revealed several sex-biased processes involved in UPR, lipid metabolism, and inflammation. We found a robust early activation of IRE1/XBP1-dependent UPR in the female ferrets. Activation of IRE1 can lead to the splicing of XBP1 mRNA (XBP1u) and subsequent expression of spliced XBP1 (XBP1s) that acts as an active and stable transcription factor for UPR-related genes (So, 2018 iScience Article 2012). Extensive studies on the role of UPR in immune cells demonstrated a fundamental impact of the IRE1/XBP1 pathway on promoting innate pro-inflammatory responses following TLR signaling in macrophages (Martinon et al., 2010) and on modulating differentiation of several cell types (reviewed in (So, 2018)), including stimulating the differentiation of B cells into antibody-secreting plasma cells (Iwakoshi et al., 2003). For example, the UPR is known to play a role in influenza virus infection (Mehrbod et al., 2019). Although predominantly observed in the lung, influenza virus infection induces ER-stress and activates the IRE1 pathway with subsequent XBP1 splicing (Hassan et al., 2012). Interestingly, emerging evidence from glycomic profiling of lung tissues collected from influenza-infected adult female ferrets indicates a link between the UPR, glycosylation, augmented immune response, and severe infection outcome; the activation of the IRE1/XBP1 pathway induces high mannose immediately upon infection, which can be recognized by the innate immune activator mannan-binding lectin (MBL2) and is associated with alveolar severity (Heindel et al., 2020). Moreover, a higher level of XBP1s expression in rat livers from prepubertal females compared with age-matched males has been reported, suggesting genetically determined female bias in XBP1s expression (Rossetti et al., 2019). Taken together, rapid activation of the IRE1/ XBP1 axis of UPR observed specifically in females, likely because of genetically encoded bias in XBP1s expression, may trigger potent immune and inflammatory responses and eventually could have contributed to a more severe disease seen in females than in males. Future studies will need to determine the functional consequences of XBP1s activation on a specific immune cell type during influenza infection and evaluate the impact of sex hormones on the pathways outlined.
Our integrative network analysis also captured two male-biased signaling pathways related to inflammatory response and lipid metabolism. Genes involved in the IL-1 pathway, including IL1RAP, JUN, IRAK3, and IRAK4, as well as two key network regulators (ACSL3 and IL18RAP), exhibited stronger inductions from 3 dpi onwards in males. ACSL3, primarily expressed in the ER and cytosolic lipid droplets, is responsible for fatty acid (FA) activation by converting free FA to FA acyl-CoA esters, an initial step contributing to lipid biosynthesis and FA oxidation (Roelands et al., 2019;Yan et al., 2015;Tang et al., 2018). Interestingly, lipid metabolism has been shown to dramatically alter the phenotype and function of monocyte/macrophages and modulate inflammatory responses, as increased lipid synthesis fueled by glucose metabolism has been linked to inflammatory macrophages, and FA oxidation is associated with alternatively activated macrophages (Remmerie and Scott, 2018;Batista-Gonzalez et al., 2020;Yan and Horng, 2020). However, the impact of ACSL3 induction and lipid metabolism on inflammatory responses in the blood cells and the crosstalk between the metabolic reprogramming and IL-1 family member IL-18-mediated signaling require further investigation. In addition to genes involved in the IL-1 pathway, we found another module enriched for the AP-1 pathway with a male-specific regulatory pattern. However, the extent of change in expression of its key regulators upon infection is similar between sexes. The AP-1 pathway composed of members in the Jun, Fos, Maf, and ATF subfamilies (Eferl and Wagner, 2003;Shaulian and Karin, 2002) plays an important role in regulating immune and inflammatory responses such as T cell activation and cytokine production (Atsaves et al., Zenz et al., 2008;Foletta et al., 1998;Schonthaler et al., 2011). Intriguingly, two key regulators in this AP-1 pathway-related module -MS4A2 acting to amplify FcεRI expression and signaling during allergic responses (Kraft et al., 2004) and HPGD responsible for inactivating potent lipid mediator PGE 2 (Tai et al., 2006) -have been implicated in susceptibility to anaphylaxis by modulating PGE 2 homeostasis in the mast cells (MCs) (Rastogi et al., 2020). PGE 2 stabilization by inhibiting HPGD-catalyzed degradation can attenuate FcεRI-mediated MC degranulation, preventing MC hyperresponsiveness and reducing susceptibility to anaphylaxis (Rastogi et al., 2020). Thus, these observations likely indicate genetically encoded male bias in the expression and regulation pattern of genes involved in specific aspects of lipid metabolism and inflammatory response in the blood cells, which requires further investigation.
Taken together, our data revealed an emerging picture in which genetic factors-mediated sex differences could manifest in the temporal dynamics and regulatory relationship of commonly induced immune and inflammatory responses, the activation of the IRE1/XBP1 pathway during the UPR, and the modulation of lipid metabolism and inflammatory response involving the IL-1 and AP-1 pathways. Although this proposed paradigm awaits further validation, especially the assessment of its generalizability in the presence of sex hormones, it provides insights into the molecular mechanism of sex differences in the cellular function and activity of blood cells, which can also be relevant to the pathogenesis of other infectious and inflammatory diseases. We noticed several limitations in this study, including the lack of granularity in differentiating cell type-specific responses and a restricted time course that does not allow a thorough investigation of adaptive immune responses, particularly humoral-mediated immunity. However, our study represents an important step toward a comprehensive ll OPEN ACCESS iScience 25, 104192, May 20, 2022 iScience Article understanding of sex differences in influenza virus-induced pathogenesis, which holds promise in developing precision medicine with improved efficacy for both sexes.

Limitation of the study
This study uses animal blood as a typical and convenient resource to analyze the host response against influenza virus. However, as a respiratory disease, the influenza virus primarily infects the respiratory system of the host. But the extraction of samples, in particular from the lung, usually requires sacrifice of the laboratory animal. A second limitation constitutes the fact that female and male ferrets were neutered because of animal welfare considerations. In particular, male non-neutered ferrets are quite aggressive against their fellow ferrets. Thus, sex hormone levels as contributing factors are lower in the studied neutered ferrets compared to the levels in non-neutered animals. However, with the neutered adult ferrets, our study provided a unique opportunity to interrogate sex-biased transcriptional response to influenza virus infection driven by genetic mediators.

STAR+METHODS
Detailed methods are provided in the online version of this paper and include the following:  (Bissel et al., 2019), adult ferrets, 6 to 12 months of age, were pair-housed in stainless steel cages (Shor-Line, Kansas City, KS) that contained Sani-Chips laboratory animal bedding (P.J. Murphy Forest Products, Montville, NJ) and provided with food and fresh water ad libitum. To achieve a mean of 20% weight loss no earlier than 8 dpi, adult ferrets were infected intranasally with the H1N1pdm09 virus at a dose of 10 6 plaque-forming units (PFU). The animals were monitored daily for the severity of clinical disease using weight loss. Disease symptoms, including elevated temperature, low activity level, sneezing, and nasal discharge, were noted if present (data not shown). Any animal losing > 20% weight loss was humanely euthanized. Blood was collected from anesthetized ferrets via the anterior vena cava after infection. The University of Georgia Institutional Animal Care and Use Committee approved all experiments, which were conducted in accordance with the NIH's Guide for the Care and Use of Laboratory Animals (Bissel et al., 2019), The Animal Welfare Act, and the Biosafety in Microbiological and Biomedical Laboratories guide of the Centers for Disease Control and Prevention and the NIH.

Ethics statement
All research studies involving the use of animals were reviewed and approved by the Institutional Animal Care and Use Committees of the University of Georgia. Studies were carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals.

Viral plaque assay
Plaque assays were performed to determine the viral burden in snap-frozen nasal washes, as previously described in (Bissel et al., 2019). Supernatants were gently thawed on ice, forced through a cell strainer (70 mm) and syringe plunger in phosphate-buffered saline, then spun down (2500 rpm, 5 minutes, 4 C) to collect the supernatant. Supernatants were diluted in Iscove's modified Dulbecco's minimum essential medium. Madin-Darby canine kidney (MDCK) cells were plated (5 3 10 5 ) in each well of a six-well plate. Samples were diluted in Iscove's modified Dulbecco's minimum essential medium (final dilution factors of 10 0 to 10 À6 ) and overlaid onto the cells in 100 mL of Dulbecco's modified Eagle's medium supplemented with penicillin-streptomycin followed by 1 hour incubation. Samples were removed, cells were washed twice, and medium was replaced with 2 ml of L15 medium plus 0.8% agarose (Cambrex, East Rutherford, NJ, USA) and incubated for 72 hours at 37 C with 5% carbon dioxide. Agarose was removed and discarded. The cells were fixed with 10% buffered formalin and then stained with 1% crystal violet for 15 minutes. After thorough washing in distilled water to remove excess crystal violet, the plates were dried, the number of plaques was counted, and the number of PFU per milliliter was calculated.

RNA-seq experiments
RNA was extracted from ferret blood using the Mouse RiboPure-Blood RNA Isolation Kit (Ambion). Both extraction methods followed the manufacturer's protocols and incorporated a DNase treatment (QIAGEN) after passing the sample through the filter cartridge. Strand-specific total RNA-seq libraries from ribosomal RNA-depleted RNA were prepared using the TruSeq Stranded Total RNA Library Prep kit (Illumina) according to the manufacturer-supplied protocol. Libraries were sequenced 100 bp paired-end to a depth of approximately 40 million host genome reads on Illumina HiSeq 2500 instruments.

RNA-seq analysis
Paired reads were aligned to the ferret genome Ensembl version 1.0.80 using tophat (version v2.0.13). Following read alignment, featureCounts (v1.4.6) was used to quantify the mRNA expression levels based on the Ensembl gene model. mRNAs with more than 5 reads in at least 1 sample were considered expressed and retained for further analysis. Otherwise, they were removed. The mRNA read counts data was normalized using the trimmed mean of M-values normalization (TMM) method of the edgeR package (Robinson et al., 2010) to adjust for sequencing library size difference.

Gene co-expression network analysis
Multiscale Embedded gene co-expression network analysis (MEGENA) (Song and Zhang, 2015) was performed to identify host modules of highly co-expressed genes in influenza infection. The MEGENA workflow comprises four major steps: 1) Fast Planar Filtered Network construction (FPFNC), 2) Multiscale Clustering Analysis (MCA), 3) Multiscale Hub Analysis (MHA), 4) and Cluster-Trait Association Analysis (CTA). The total relevance of each module to influenza virus infection was calculated by using the Product of Rank method with the combined enrichment of the differentially expressed gene (DEG) signatures as implemented: G j = Q i g ji , where, g ji is the relevance of a consensus j to a signature i; and g ji is defined as ðmax j ðr ji Þ + 1 À r ji Þ= P j r ji , where r ji is the ranking order of the significance level of the overlap between module j and the signature.

Identification of enriched pathways and key regulators in the host transcriptome modules
To functionally annotate gene signatures and gene modules derived from the host transcriptome data, we performed an enrichment analysis of the established pathways and signatures-including the MSigDB and ARCHS 4 tissues (Lachmann et al., 2018). The hub genes in each subnetwork were identified using the adopted Fisher's inverse Chi-square approach in MEGENA; Bonferroni-corrected p-values smaller than 0.05 were set as the threshold to identify significant hubs.

Correlation analysis
The correlation between modules and physiological traits was performed using Spearman's correlation. The correlation between the FCs of DEGs in females and males over time was estimated using Pearson's correlation with the Benjamini-Hochberg correction.

Similarity comparison of modules and their key drivers
The pairwise similarity between a given female module and every male module or between their key drivers was estimated using Fisher's Exact Test (FET) with Bonferroni correction. Modules in one sex without counterparts in the other sex (FET p % 0.05) were considered sex-unique module compositions. Differential Gene Correlation Analysis (DGCA) was also performed with the ''moduleDC'' function implemented in the DGCA package (McKenzie et al., 2016) to further determine the unique regulatory relationship of the overlapped gene members between a pair of similar modules in females and males. Modules with significant differential correlation (p % 0.05) with all of their counterparts were considered to exhibit sex-specific regulatory relationships.


Loss of Ecrg4 improves calcium oxalate nephropathy

Kidney stone is one of the most frequent urinary tract diseases, affecting 10% of the population and displaying a high recurrence rate. Kidney stones are the result of salt supersaturation, including calcium and oxalate. We have previously identified Esophageal cancer-related gene 4 (Ecrg4) as being modulated by hypercalciuria. Ecrg4 was initially described as a tumor suppressor gene in the esophagus. Lately, it was shown to be involved as well in apoptosis, cell senescence, cell migration, inflammation and cell responsiveness to chemotherapy. To the best of our knowledge, nothing is known about ECRG4’s function in the renal tissue and its relationship with calciuria. We hypothesized that the increased expression of Ecrg4 mRNA is triggered by hypercalciuria and might modulate intratubular calcium-oxalate precipitation. In this study, we have first (i) validated the increased Ecrg4 mRNA in several types of hypercalciuric mouse models, then (ii) described the Ecrg4 mRNA expression along the nephron and (iii) assessed ECRG4’s putative role in calcium oxalate nephropathy. For this, Ecrg4 KO mice were challenged with a kidney stone-inducing diet, rich in calcium and oxalate precursor. Taken together, our study demonstrates that Ecrg4’s expression is restricted mainly to the distal part of the nephron and that the Ecrg4 KO mice develop less signs of tubular obstruction and less calcium-oxalate deposits. This promotes Ecrg4 as a modulator of renal crystallization and may open the way to new therapeutic possibilities against calcium oxalate nephropathy.

Introduction
Prevalence of chronic kidney diseases and of kidney stones is constantly increasing [1]. In both conditions, supersaturation of calcium-oxalate salts and crystal formation may result either in obstruction of renal tubules or in stone formation. Thus, hypercalciuria and hyperoxaluria are two major risk factors for crystallogenesis in urine [2]. We have previously identified the Ecrg4 gene as upregulated in hypercalciuric mice. Highly conserved from fish to humans, Ecrg4 encodes a 148 amino acids precursor protein that can be processed into various peptides, without identified regulatory proteases [3]. Ecrg4 mRNA can be found in various tissues, such as brain, esophagus, heart and kidney, whereas, the protein ECRG4 was documented in the heart and adrenal gland [4]. The functions of ECRG4 and derived peptides are still largely unknown. It was first speculated that Ecrg4 might be a tumor suppressor gene, due to its decreased expression in esophageal cell carcinomas, while still detectable in the normal esophageal epithelia surrounding the tumor [5]. Yet, in vivo studies have extended the anti-tumoral role of Ecrg4 to glioma and to prostate, colorectal and breast cancer [6][7][8]. Further, Ecrg4's overexpression was shown to affect apoptosis, cell senescence, cell migration, inflammation and cell responsiveness to chemotherapy [3]. Its activity depends on its cellular localization, secretion and processing, which argues for a non-traditional tumor suppressor action, and rather for a role close to a cytokine/chemokine. Downstream genes that were shown to be activated by Ecrg4 include Nfkb1, Nfkb2, P53 and Cox2 [3].
Ecrg4's role in the kidney and especially in hypercalciuria is unknown. Due to its higher expression in a hypercalciuric mouse model, we hypothesized that Ecrg4 mRNA is triggered by hypercalciuria and might modulate kidney stone formation. In the present work, we first describe Ecrg4 mRNA expression in the kidney. Second, we show that Ecrg4 expression is increased in various hypercalciuric mouse models. Third, we assessed the potential role of Ecrg4 in kidney stone formation by challenging Ecrg4 KO and control mice with a diet inducing hypercalciuria and hyperoxaluria. We found substantial protection against calcium oxalate nephropathy in male mice devoid of Ecrg4. In total, this piece of data points towards Ecrg4 as a potential regulator of calciuria and stone formation.

Animals
The State veterinarian Office (Office vétérinaire cantonal, Canton de Vaud, Switzerland) approved all animal studies (authorization numbers VD3261 and VD2987). All breeding and cohort colonies were hosted in our animal facility under approved protocols. Mice were housed four to five per cage, with free access to water and food (standard mouse diet #3800, KLIBA, Kaiseraugst, Switzerland), in a temperature and humidity-controlled room with an automatic 12/12-h light/dark cycle. Heterozygous Ecrg4 mice were purchased from the Mutant Mouse Regional Resource Center (B6;129S5-1500015O10Rik tm1Lex /Mmucd). In this model, coding exon 1 of the RIKEN cDNA 1500015O10 gene was targeted by homologous recombination. Mice were backcrossed for 10 generations and heterozygous males and females were bred in order to obtain Ecrg4 -/-(KO) mice. Littermates Ecrg4 +/+ have been used as controls for all experiments. Kidney-specific sodium-calcium exchanger 1 (NCX1) Ncx1-knockout mice (Pax8-LC1-Cre NCX1fl/fl) were also used in the study (unpublished, manuscript in preparation). Claudin-2 (Cldn2) KO mice (B6; 129S5-Cldn2 tm1Lex /Mmucd) were purchased from Mutant Mouse Regional Resource center.

Microdissection of nephron segments
Mice were deeply anesthetized by intraperitoneal injection with Ketanarkon (Streuli Pharma AG, Uznach, Switzerland; 100 μg/g body weight) and Rompun (Bayer, Leverkusen, Germany; 10 μg/g body weight), and the left kidney was perfused first with PBS, then with DMEM/F12 (1:1, Life Technologies, Carlsbad, USA) supplemented with 40 mg/ml of Liberase Blendzyme 2 (Roche, Basel, Switzerland). Thin slices of the kidney were cut along the corticomedullary axis and incubated 40-60 min at 37˚C in the same DMEM/F12/Liberase medium. Then, the kidney slices were transferred into DMEM/F12 (1:1) alone to stop the digestion, and the microdissection was performed under a magnifier in ice-cold 0.05%BSA/DMEM/F12. The proximal convoluted tubule (PCT), the thick ascending limb (TAL), distal convoluted tubule and cortical connecting tubule (DCT-CNT), and the cortical collecting duct (CD) were isolated based on their specific morphology.

RNA extraction and quantitative PCR
Microdissected tubules. Immediately after microdissection, tubules were subjected to RNA extraction using the RNeasy Micro Kit (# 74004, Qiagen, Hilden, Germany) according to the manufacturer instructions.
Other tissues. RNA from kidneys of mice injected with 1,25(OH) 2 -vitamin D and PTH as previously described [9], was used to assess the Ecrg4 expression. Tissues were homogenized in TRIzol 1 Reagent solution (#15596026 Thermo Fisher, Waltham, USA) followed by extraction with 1-bromo-3-chloropropane reagent (BCP, Molecular Research Center, Cincinnati, USA) and isopropanol precipitation. RNA (1 μg) was reverse transcribed using PrimeScript RT reagent kit (#RR037B Takara Biotechnology, Otsu, Japan) according to manufacturer's guidelines. TaqMan Gene Expression Assays (#4370074, Applied Biosystems, Warrington,UK) was used to detect Ecrg4 (Mm00470447_m1), together with Actb (Mm4351315_m1). Other genes were quantified using SYBR green PCR master Mix (#4367659, Applied Biosystems, Warrington,UK). The primers used are listed in Table 1. Primers were ordered from Microsynth (Balgach, Switzerland). Quantitative real-time PCRs were carried out on an ABI PRISM 7500 equipment (Applied Biosystems, Warrington,UK). If the cycle threshold (CT) were >36, the expression was considered as null. The relative expression of the genes was calculated using the comparative 2 −ΔΔCT method.

Metabolic and endocrine studies
Male and female mice aged 6 weeks were housed individually in metabolic cages (Tecniplast, Buguggiate, Italy) for 2 days in order to be habituated before baseline urine collection, with free access to water and food. After the habituation period, control and Ecrg4 KO mice were fed either the chow diet or the crystal forming diet CaOx (1.5% Calcium Chloride and 2% Hydroxyl-L-proline) for 8 days. Twenty-four hour urine collection was repeated on day 1 and day 8, by housing the mice in metabolic cages.
Urine sodium and potassium concentrations were determined by flame photometry (Instrumentation laboratory), while the urine creatinine, calcium and magnesium concentrations were measured in the Laboratoire Central de Chimie Clinique, Lausanne University Hospital (Lausanne, Switzerland). Urine osmolality was measured with an automated freezing point osmometer (Advanced model 2020 Multi-Sample Osmometer, Advanced Instruments, Norwood, USA) and the urine pH using a pH-meter (#6.0224.100, Metrohm, Herisau, Switzerland). The Laboratoire Central de Chimie Clinique, Lausanne University Hospital (Lausanne, Switzerland), measured all the blood parameters unless otherwise specified. Creatinine concentration was measured using the enzymatic method.
For the dihydrotachysterol experiment, the chow diet of control male mice was supplemented with 1.5 mg/kg dihydrotachysterol (D9257, Merk, Kenilworth, USA) during 7 consecutive days.
For the analysis of organs from PTH-treated animals, material harvested from a previous study was used [9]. In this study, male 12-13 weeks old C57BL/6N mice received a subcutaneous injection of 80 μg/kg body weight human PTH 1-34 (#P3796, Sigma Aldrich, St. Louis, USA) dissolved in NaCl 0.9% or NaCl 0.9% alone as vehicle. Animals were dissected 2 hours after injection.
For the acute furosemide experiment, male C57BL/6N mice aged 12 weeks received a single intraperitoneal injection (Lasix, Sanofi-Aventis, Switzerland; 20mg/kg body weight) diluted in saline solution. The control group received an intraperitoneal injection of NaCl 0.9%. The mice were sacrificed after 15min, 30min, 60min or 240min and the kidneys were immediately harvested.
For the chronic furosemide experiment, male C57BL/6N mice aged 12 weeks received a daily intraperitoneal injection for 6 days of either furosemide diluted in saline solution (20mg/ kg body weight), or saline solution for the control mice. The mice were sacrificed at day 7 and the kidneys were harvested.

Kidney histology and crystals
Left kidneys were fixed in 4% paraformaldehyde, embedded in paraffin and 5μm sections were cut. Sections were then stained with Pizzolato and H&E. Images were taken on Leica DMi8 microscope. To determine the area of calcium oxalate crystals (stained in black by Pizzolato) over the kidney's surface area, images were obtained at x10, and analyzed with ImageJ. The result was expressed as percentage of black area over the whole kidney area. Similarly, ImageJ software was used for the analysis of tubular dilatation on H&E-stained kidney sections. The investigator was blinded of the code when analysis were performed.

Immunohistochemistry
Organs were fixed in 4% paraformaldehyde in Phosphate Buffer Solution and then embedded in paraffin before being cut as 5μm-thick sections. Sections were first hydrated gradually through decreasing concentrations of ethanol and then washed in deionized water. Antigen unmasking was done in 10mM sodium citrate buffer, pH 6.0, for 1h at 96˚C, followed by incubation in 0.3% hydrogen peroxide for 10 min at room temperature, and by 1h in blocking buffer (NP-40 0.5%, BSA 2%, normal goat serum (NGS) 3% in PBS). Sections were incubated with primary antibodies for 1h at room temperature. Primary antibodies were washed in PBS three times for 5 min, followed by incubation with anti-mouse or anti-rabbit serum as appropriate for 30 min and developed in 3,3'-diaminobenzidine (DAB substrate kit, #ab64238, Abcam, Cambridge, UK). Lastly, they were counterstained with Nuclear Fast Red Solution (6409-77-4, Merck, Kenilworth, USA).

Immunofluorescence
Deeply anesthetized mice were perfused via cardiac puncture with 4% paraformaldehyde in PBS. Harvested organs were incubated in 30% sucrose in PBS for at least 24 h before being embedded in Tissue-Tek OCT compound (#4583,Sakura Finetek, Alphen aan den Rijn, The Netherlands) and cut as 7μm-thick sections. Sections were incubated 1 h with blocking buffer (NP-40 0.5%, BSA 2%, normal goat serum (NGS) 3% in PBS) at room temperature, followed by primary antibody incubation overnight at 4˚C. After washing three times with PBS, sections were incubated 1 h at room temperature with secondary antibodies and then washed four times with PBS. The sections were then mounted using Fluoromount-G mounting medium (#0100-01, Southern Biotech, Birmingham, USA). Fluorescent images were visualized using a laser scanning confocal microscope (SP5 AOBS Confocal Microscope, Leica Microsystems, Wetzlar, Germany).

Statistical analyses
Comparisons between groups were conducted using GraphPad Prism software (version 9.0.0). Results are presented, as means ± SD, unless otherwise stated. Statistical analyses were performed using Student's t tests (2-tailed), one-way ANOVA, or two-way ANOVA, using Tukey correction for multiple comparison. Values of p < 0.05 were considered statistically significant.

Ecrg4 is expressed in the kidney and along the distal part of the nephron
The expression of Ecrg4 mRNA in male kidney was analyzed and compared to other organs, including adrenal glands, brain, heart and esophagus. Ecrg4 mRNA was detected in all tissues tested ( Fig 1A). Female kidney and heart tissues have an increased Ecrg4 expression compared to male mice (S1 Fig). Substantial efforts have been done to identify ECRG4 protein in the same tissues by Western blot or immunostaining. However, none of the antibodies showed specificity in the tested mouse tissues when compared to the same tissues obtained from Ecrg4 KO mice (S2-S7 Figs).
After confirming the presence of Ecrg4 mRNA in extracts obtained from the whole kidney, we looked whether Ecrg4 expression was restricted to certain segmental parts of the nephron or if it was ubiquitous. We microdissected the tubular segments from the kidney of wildtype mice and found that Ecrg4's presence was limited to the thick ascending limb (TAL), distal convoluted tubule/connecting tubule (DCT/CNT) and cortical collecting duct (CD) segments, while no expression was found in the proximal (PROX) tubule ( Fig 1B).

Ecrg4 transcript is upregulated in hypercalciuric mouse models
We identified Ecrg4 as an upregulated gene in a RNA-sequencing screen of kidneys from mice devoid of the sodium-calcium exchanger NCX1 and which are hypercalciuric. We wondered whether Ecrg4 mRNA levels would be increased in other models of hypercalciuria. We analyzed Ecrg4's expression, compared to control littermates in kidney from Ncx1-kidney-specific KO mice, Claudin-2 (Cldn2) KO mice and mice fed the vitamin D analog, dihydrotachysterol (DHTS). Ncx1-kidney-specific KO mice and Cldn2 KO mice are genetic mouse models of hypercalciuria due to a primary defect in renal tubular (respectively DCT and proximal tubule) calcium transport [11]. DHTS, the vitamin D analogue, is a pharmacological model due to increased intestinal absorption, plasma Ca 2+ levels and urinary Ca 2+ excretion [12]. Ecrg4 mRNA expression was substantially increased in these three chronic hypercalciuric mouse models, as depicted in Fig 2A. We also tested the effect of furosemide-which induces hypercalciuria by blocking calcium reabsorption in the thick ascending limb of Henle-for six consecutive days. No effect of furosemide on Ecrg4 mRNA level was observed. Considering a possible tubular adaptation to hypercalciuria in the case of chronic furosemide treatment, we checked for renal Ecrg4 mRNA expression after single furosemide treatment. Again, we did not find any change in Ecrg4's expression after acute furosemide, compared to vehicle, up to 240 minutes after furosemide injection (Fig 2B). Hypercalciuria was however substantial only after 15min following the furosemide administration (S8 Fig). Next, we checked if renal Ecrg4's expression was influenced by the calcitropic hormones 1,25(OH) 2 -vitamin D and PTH under acute stimulating conditions. No effect of these hormones on the renal Ecrg4 mRNA level was observed (Fig 2C).
After having observed a response of Ecrg4's expression to chronically induced hypercalciuria, we wondered if the increase in gene expression was segment-specific or was concerning all tubular segment in which Ecrg4 was found to be expressed. We microdissected the renal tubules of the hypercalciuric kidney-specific Ncx1 KO mice and found that Ecrg4 is significantly increased in the TAL, DCT-CNT and CD but not in the proximal tubule compared to control littermates ( Fig 2D).
In total, we found that Ecrg4 expression is increased in the kidneys of several chronic hypercalciuric mouse models and concerns all segments in which Ecrg4 was found to be expressed, namely TAL, DCT and CD.

Ecrg4 KO male mice, but not females, are resistant to induced hypercalciuria
In order to get more insights into the role of Ecrg4 in calcium handling by the kidney, we obtained Ecrg4 KO mice and backcrossed them in the C57BL/6 background (Fig 3A). No obvious phenotype (fertility, weight, size, behavior, macroscopic appearance and fur) was observed in KO mice compared to littermate controls. Similarly, no difference in the tested blood and urine parameters was observed (Tables 2 and 3).
To further test the hypothesis that Ecrg4 modulates calcium-oxalate crystal nephropathy, we fed the mice a diet enriched in calcium (1.5%) and in the oxalate precursor, hydroxyproline

PLOS ONE
Role of Ecrg4 in the kidney (2%), further referred to as the CaOx diet (Fig 3B). Ecrg4 KO male mice fed the CaOx diet had a significantly lower fractional excretion of calcium compared to control mice fed the same diet, despite comparable calcemia and blood creatinine values ( Table 2). Such finding was not observed in females (Table 3). Under the CaOx diet, urinary calcium/creatinine ratio was increasing between baseline and day 8 (Fig 3C). Interestingly, after 8 days of treatment, urinary calcium excretion of Ecrg4 KO male mice was significantly lower compared to control male mice fed the same diet ( Fig 3C). As differences of sodium excretion was observed between the groups under CaOx diet (Fig 3D), the urinary calcium/sodium ratio was calculated ( Fig 3E) and showed that the lower calciuria observed in Ecrg4 KO mice is independent of sodium. Female data are provided in supplemental material, but did not show difference in renal calcium handling between Ecrg4 KO and controls (S9-S11 Figs). Altogether, this data shows that Ecrg4 KO male mice have lower fractional excretion and lower calciuria than controls while exposed to the CaOx diet.

Ecrg4 KO male mice, but not females, have less renal tubular obstruction under CaOx diet
Control male mice exposed to the CaOx crystal forming diet exhibited intraluminal crystal deposits, clearly detected by Pizzolato staining (Fig 4A) already after 8 days of treatment. The crystals were localized predominantly in the papilla, with fewer crystals observed in the kidney cortex or medulla.
In line with the observed decrease in calcium excretion, Ecrg4 KO male mice developed less intratubular deposits compared to control mice, with an important inter-individual variability ( Fig 4C). Moreover, the tubules were significantly less dilated in Ecrg4 KO mice compared to control animals (Fig 4B and 4D). Of note, and as often observed in the field, females were completely resistant to crystal formation (S12 Fig). Overall, male mice devoid of Ecrg4 had less crystal deposits and less tubular dilation, indicative of less tubular plugging.
In order to get more insights into calcium handling by the kidneys of Ecrg4 KO mice, mRNA expression levels of genes involved in transepithelial calcium transport or its regulation were assessed. No change in Slc9A3, Pthr1, Cldn14, Cldn2, Cldn16, Atp2b4, Trpv5, Calb1,

PLOS ONE
Role of Ecrg4 in the kidney Slc8A1, Aqp2, Pvalb, Casr, Vdr was found between the kidneys of Ecrg4 KO and control mice exposed to the CaOx diet (S13 Fig). However, a marked increase of PTHR1 can be seen in the kidney tissue of Ecrg4 KO mice compared to controls (S14 Fig). In addition, genes involved in Ecrg4-dependent downstream pathways (Nfkb1, Nfkb2, P53 and Cox2) were unchanged, with the notable exception of P53, which was less expressed in the KO mice (S15A Fig). Finally, we exposed mouse cortical collecting duct cells (mCCD) to different calcium oxalate crystal concentrations (0, 300 or 600 ug of crystal/ml) and Ecrg4 mRNA level was measured by qPCR (S15B Fig). We observed a dose-dependent decrease of Ecrg4 mRNA upon treatment with higher crystal concentrations, as well as a decrease in Nfkb1 expression when cells were exposed to the highest calcium oxalate crystal concentration (S15C Fig). No other change in the expression levels of the Ecrg4 downstream genes (Nfkb1, Nfkb2, P53 and Cox2) was observed.

Discussion
This study investigated the expression of Ecrg4 in the renal tissue, its regulation and its potential role in the development of kidney stones. We found expression of Ecrg4 along the distal tubules, showed upregulation of Ecrg4 mRNA expression in several chronic hypercalciuric mouse models and showed less signs of tubular obstruction in Ecrg4 KO mice fed a calciumoxalate rich diet. This promotes Ecrg4 as a modulator of renal crystallization.

Ecrg4's expression in renal tissue
We detected the presence of Ecrg4 mRNA in the distal segments of the nephron (TAL, DCT/ CNT and CD), while the proximal tubule was negative for Ecrg4's expression, in line with a recently published mouse single-cell RNA-seq analysis [13].
We spent substantial energy to detect and quantify ECRG4's protein expression in the kidney and other organs (S2-S7 Figs), but were unsuccessful to detect any specific signal. This could be explained by the fact that the specificity of the commercial antibodies used in this study has been tested only a minima by the providing companies, usually only in cell lines overexpressing ECRG4, and were not validated in native tissues obtained from control and Ecrg4 KO mice. An alternative explanation for the lack of detection might be that ECRG4 is processed very quickly in vivo or that posttranslational modifications might prevent its detection. Indeed, the 148 amino acids full-length protein was shown to be cleaved into at least eight peptides of varying molecular weight, ranging from~17 kDa to 2 kDa [14]. This process might render ECRG4's detection difficult with antibodies developed against full-length ECRG4. We thus looked for alternative methods that could help us identify the ECRG4 protein. A mass spectrometry analysis was performed on various protein extracts, from heart, brain, and Ecrg4-transfected HEK cells (S1 and S2 Tables). However, the detection level of ECRG4 fragments was discouragingly low, even in the overexpressing HEK cell system. We thus concluded that the ECRG4 protein expression is too low to be identified by the available tools or that only fragments of ECRG4 should be looked for. We thus continued our study only by interpreting the mRNA level of expression of Ecrg4.

Ecrg4 is regulated in models of hypercalciuria
We have previously identified Ecrg4 as an mRNA upregulated in the hypercalciuric kidneyspecific Ncx1 KO mouse model. Here, we confirmed that Ecrg4 expression is higher in the kidney of Ncx1 KO mice compared to controls in all tubular segments expressing Ecrg4. We looked whether this regulation was observed in other models of hypercalciuria. We used the hypercalciuric Cldn2 KO mouse model [11], lacking proximal tubule calcium reabsorption, and mice fed the vitamin D analog, DHTS, which causes hypercalcemia and hypercalciuria. In these two models, up to 2.5 times increase in renal Ecrg4 mRNA expression was observed compared to controls. By contrast, no change in the Ecrg4 level was found when mice were injected acutely or for six consecutive days with furosemide, a known inducer of calciuria. We speculate that this discrepancy might be due to the relative short term and transient hypercalciuria induced by furosemide compared to chronic and sustained hypercalciuria of the other models. Thus, we propose that Ecrg4's increased expression is triggered only by chronic and sustained hypercalciuria.
The mechanism by which Ecrg4 is regulated by urine calcium remained undetermined. We explored whether the calcitropic hormones, PTH or 1,25(OH) 2 -vitamin D, might have a direct effect on Ecrg4's expression. We did not observe any change in Ecrg4 renal expression after short term exposure to the two hormones. This might be due to the relative short stimulation time (2h for PTH and 6h for 1,25(OH) 2 -vitamin D), even if Cyp24a1 and Cyp27b1 transcript expression, used as controls, showed substantial increase after exposure to either of these hormones [9]. As previously noted, long-term exposure to the vitamin D analog, DHTS, induced a strong Ecrg4 upregulation.
Based on this piece of data, we conclude that Ecrg4 is regulated by long term exposure to sustained hypercalciuria, with no direct short-term effect of furosemide, PTH or 1,25(OH) 2vitamin D. The precise mechanism by which tubular calcium regulates Ecrg4 expression remains to be identified.

Loss of Ecrg4 protects against tubular calcifications in male mice
We used a diet rich in calcium and in the oxalate precursor, hydroxyproline, for 8 days to induce hypercalciuria, hyperoxaluria, and calcium-oxalate nephropathy. In agreement with previous studies [15], we showed that kidneys of male mice developed a CaOx crystal nephropathy, while females were resistant, despite similar high urinary calcium excretion. Of note, sex differences in stone formation have been observed in humans and were attributed to sex hormones [16][17][18][19][20]. These differences seem to be more pronounced in mice and will need further studies to be deciphered. On the same note, our results show a gender-specific difference in Ecrg4's expression in the renal tissue, under control conditions (S1 Fig). Consistent with previously published evidences, increased Ecrg4 expression inhibits cell proliferation [21], which was found to account for renal crystal deposits in the setting of supersaturated urines [22,23]. Further studies should assess if the difference in Ecrg4 levels is directly involved in the different outcome in calcium-oxalate nephropathy between male and female mice.
To our surprise, we found that male mice devoid of Ecrg4 and exposed to the CaOx diet had less tubular dilation and intra-renal calcifications. In these mice, we also found a preserved renal function, indicating that overall the mice were protected from tubular obstruction by calcium oxalate crystals. This protection could be due to the role of Ecrg4 in the kidney or in other organs. The whole body constitutive knockout mouse model we used did not allow us to discriminate between the two possibilities. However, some observations suggest that the loss of Ecrg4 may play a protective role by both renal and extra-renal effects. Hence, when exposed to the CaOx diet, control male mice had increased calcemia and calciuria compared to Ecrg4 KO male mice. This suggests that Ecrg4 KO mice adapt to the CaOx challenge by controlling their calcemia and calciuria by an unknown mechanism, potentially by decreasing their intestinal calcium absorption. Moreover, Ecrg4 KO mice displayed lower fractional excretion of calcium, indicative of a direct effect of ECRG4 on the tubular calcium reabsorption. In an attempt to understand the mechanisms by which Ecrg4 KO mice might have a different renal calcium handling, we analyzed the mRNA expression (S13 Fig), as well as protein level (S14 Fig) of several renal genes and proteins involved in calcium homeostasis. We found similar levels between KO and control mice for all the tested candidates, except for the PTHR1 which was significantly increased in the Ecrg4 KO mice. In these mice, this might indicate a possible adaptive mechanism to regulate calcium homeostasis that involves PTH/PTHR1 signaling pathway in the distal segment of the nephron.
Altogether, systemic as well as renal mechanisms could concur to alleviate crystal formation in Ecrg4 KO mice under enriched calcium and hydroxyproline diet.
As Ecrg4 is a tumor suppressor gene involved in the p53 and NFKB pathways [24][25][26], we wondered whether Ecrg4 might be involved in mediating inflammation or tubular cell renewal, explaining the attenuated damages seen in the kidneys of Ecrg4 KO mice. First, we found decreased expression of p53 mRNA in the kidneys of Ecrg4 KO mice (S15A Fig). This suggests that under the setting of CaOx diet challenge, male mice might have more cellular proliferative capacity to cope with crystal-induced injuries. Second, we found a down-regulation of the Nfkb1 gene in the mouse cortical collecting duct cell line (mCCD cells) exposed to calciumoxalate monohydrate crystals (S15B and S15C Fig). In this system, the decrease of Nfkb1 expression might indicate that crystal-induced inflammation is reduced in the absence of Ecrg4. Indeed, it was previously shown that ECRG4 peptide induces the expression of proinflammatory factors via a variety of intracellular signaling pathways. For example, increased reactive oxygen species levels, activated NFkB signaling pathway and interaction with the innate immunity receptor complex TLR4/CD14/MD2 complex, was demonstrated upon ECRG4 binding to LOX-1, and other scavenger receptors, such as Sccarf1, Cd36 and Stabili-1 [27,28]. Altogether, deletion of Ecrg4 might limit the defense mechanisms against crystalinduced injuries. However, how cells sense hypercalciuria and increase Ecrg4 expression still remains to be identified.

Strengths and limitations
We are not aware of any other study addressing the role of Ecrg4 in the kidney and showing that Ecrg4 is involved in renal calcium handling and in the risk of calcification. However, several limitations warrant mention. First, our evidence is mainly based on mRNA expression levels. As discussed previously, we were not able to detect ECRG4 full-length protein or fragments with the available anti-ECRG4 antibodies. Second, the mechanisms by which loss of Ecrg4 reduces tubular calcifications remain open. We observed lower calciuria in male mice under CaOx diet compared to controls, but whether this might be sufficient to decrease intratubular crystal formation or retention remains unsolved. Finally, further studies will need to examine whether the limited renal insufficiency and hypercalciuria in the Ecrg4 KO mice is rather time dependent, or if there is a long-term adaptation mechanism, which might involve some other organs than the kidney.
By showing that Ecrg4 is involved in urine calcium handling and crystal nephropathy, we hope to open new perspectives of research in the field. water intake parameters were measured over 24h period for males and females. At baseline, all mice were fed the chow diet and then exposed to either CaOx diet or chow diet (n = 4-5). Data are shown as mean ± SD. p value was calculated using two-way ANOVA with Tukey correction for multiple comparison. Stars indicate � p<0.05 between the control and Ecrg4 KO, § indicates p<0.05 between the controls of different diets, # indicates p<0.05 between the Ecrg4 KO of different diets. (TIF) S11 Fig. 24h urinary parameters. Urinary phosphate (A), potassium (B) excretion, osmolality (C) and pH (D). Mice were individually housed in metabolic cages, after exposure to chow or CaOx diet (n = 4-5). Data are shown as mean ± SD. p value was calculated using two-way ANOVA with Tukey correction for multiple comparison. Stars indicate � p<0.05 between the control and Ecrg4 KO, § indicates p<0.05 between the controls of different diets, # indicates p<0.05 between the Ecrg4 KO of different diets. Mice were fed either the chow diet or challenged with the CaOx diet. Western blot analysis of (B) PTHR1 protein in control vs Ecrg4 KO male mice kidney tissue (n = 4-5). Mice were challenged with the CaOx diet. Quantities represented by the gel bands are expressed as intensity relative to β-Actin. Quantities represented by the gel bands are expressed as intensity relative to β-actin. All relative intensity results are presented as the means ± SD. Stars indicates � p<0.05. P values are calculated using two-way ANOVA test, with Sidak's correction for multiple comparison (A) or Student t-test (B). (TIF)

S15 Fig. mCCD cells stimulated with CaOx monohydrate crystals. (A)
The downstream genes that are activated upon ECRG4 signaling were quantified by qPCR in kidney extracts from control and Ecrg4 KO male mice. Stars indicates � p<0.05. P values are calculated using Student t-test (n = 4). (B) mCCD cells were incubated with CaOx monohydrate crystals for 16h. The Ecrg4 mRNA expression is shown as response to different crystals concentrations. (C) The downstream genes that are activated upon ECRG4 signaling were quantified in the mCCD cells upon calcium-oxalate monohydrate crystals incubation. Stars indicates � p<0.05. P values are calculated using one-way ANOVA comparing the mean of each CaOx crystal dosage (300 and 600 ug/ml CaOx) with the mean of the control column at 0 ug/ml CaOx crystal dosage (n = 3). Data are shown as mean ± SD. (TIF) S1 Table. Identified proteins by mass spectrometry in control heart and brain tissues.